Abstract Delayed Graft Function (DGF) refers to failure of a kidney to function optimally after transplantation. DGF is a serious clinical problem that independently predicts reduced 1- and 5- year kidney transplant survival. Prolonged Cold Ischemia (CI) of > 24 hours is a well-known risk factor for DGF, but the mechanism by which DGF occurs is not known. This proposal endeavors to determine how prolonged CI predisposes to DGF. Our published data demonstrate that mouse and porcine kidneys subjected to prolonged CI have increased caspase-3 and renal tubular epithelial cell (RTEC) apoptosis. We have developed a mouse kidney transplant model of DGF that demonstrates: (a) Prolonged CI alone results in RTEC apoptosis; (b) Prolonged CI followed by kidney transplant results in DGF, manifested by increased serum creatinine (sCr.), RTEC apoptosis and tubular necrosis (ATN); (c) Kidney transplant alone without CI results in neither RTEC apoptosis, ATN, nor DGF. Thus we propose to demonstrate the mechanisms by which RTEC apoptosis during prolonged CI predisposes to ATN and DGF. Our preliminary data implicate X-linked inhibitor of apoptosis (XIAP) protein in caspase-3 mediated RTEC apoptosis during prolonged CI. Our preliminary data also demonstrate that prolonged CI of mouse RTEC in vivo and in vitro results in decreased XIAP, apoptosis, and release of high-mobility group protein 1 (HMGB1), a ligand of Toll-like receptor 4 (TLR4). HMGB1 binding of TLR4 activates RIP kinases - 1 and -3, which are serine/threonine protein kinases essential for cellular programmed necrosis. Thus, our overall hypothesis is that before kidney transplant, prolonged CI leads to RTEC apoptosis and release of HMGB1 from apoptotic nuclei to the cytoplasm, and into the interstitial space. We also hypothesize that after transplant of kidneys subjected to prolonged CI: (a) there is increased RTEC expression of TLR4; (b) TLR4 is activated by HMGB1; (c) TLR4 subsequently activates RIP kinases - 1 and -3 leading to ATN of other RTECs and DGF. In Specific Aim 1, we will determine whether upregulation or inhibition of XIAP has an effect on RTEC apoptosis, ATN, and sCr. in a mouse model of DGF. XIAP inhibition will be achieved by using either siRNA against XIAP or XIAP deficient mice. XIAP overexpression will be achieved by inhibition of HTRA2, a protein released from disrupted mitochondria that prevents the association of XIAP with caspase-3, thus causing RTEC apoptosis. In Specific Aim 2, we will determine whether upregulation or inhibition of HMGB1 has an effect on ATN and sCr. in a mouse model of DGF. HMGB1 inhibition will be achieved using HMGB1 neutralizing antibody. HMGB1 overexpression will be achieved using recombinant HMGB1. In Specific Aim 3 we will determine whether upregulation or inhibition of TLR4 has an effect on ATN and sCr.in a mouse model of DGF. TLR4 overexpression will be examined in wild-type kidneys transplanted after prolonged CI, and wild-type kidneys transplanted into TLR4 deficient syngeneic recipients. TLR4 inhibition will be achieved by; (a) an inhibitory antibody against TLR4; (b) transplanting TLR4 deficient kidneys into wild-type syngeneic recipients.